Fungal mitochondria govern both gliotoxin biosynthesis and self-protection.

Gliotoxin (GT) is a potent epipolythiodioxopiperazine toxin produced by the opportunistic pathogen Aspergillus fumigatus that contributes to virulence and inhibits competing microorganisms. However, GT is highly toxic to the producer itself, necessitating robust self-protection mechanisms. Here, we used a comparative transcriptomics approach between A. fumigatus (GT producer) and A. nidulans (non-producer) to identify additional genetic determinants of GT self-protection downstream of the transcription factor RglT. We characterized five RglT-dependent genes: abcC1 (ABC transporter), mfsD (major facilitator superfamily transporter), oxrA (oxidoreductase), mtrA (putative methyltransferase), and nmrC (a GATA-type repressor). Deletion mutants in A. fumigatus and A. nidulans revealed that all except oxrA were required for full GT protection, with ΔmtrA and ΔnmrC exhibiting distinct phenotypes in oxidative stress and iron-starvation conditions. Transcriptomic profiling and protein network analysis showed that MtrA and NmrC influence mitochondrial functions, particularly ubiquinone biosynthesis, despite not localizing to mitochondria. Functional assays confirmed that GT exposure disrupts mitochondrial integrity and sensitizes A. fumigatus to mitochondrial inhibitors. Notably, GT-induced cell death was associated with mitochondrial fragmentation but lacked hallmarks of apoptosis-like nuclear damage. Together, our findings reveal new genetic components of GT detoxification and establish a critical role for mitochondrial function in A. fumigatus GT self-protection and production.IMPORTANCEGliotoxin (GT) plays a central role in the pathogenicity of Aspergillus fumigatus by enabling immune evasion and microbial competition, but its extreme toxicity also threatens the fungus itself. Although core GT biosynthetic and detoxification mechanisms are well studied, the full genetic network safeguarding against GT's effects remains incompletely understood. This study identifies new RglT-regulated genes that contribute to GT self-protection and demonstrates that mitochondrial function is crucial for surviving GT exposure. Remarkably, similar protective pathways are active in both GT-producing and non-producing fungi, underscoring the ecological relevance of GT defense mechanisms. These findings deepen our understanding of fungal toxin tolerance and highlight mitochondria as a potential vulnerability that could be exploited for antifungal interventions.